Preterm birth is the leading cause of infant mortality, yet we know little of human myometrial signals that initiate of labor contractions in normal-length pregnancies, let alone preterm pregnancies. Benign tumors of the myometrium, leiomyomas, are found in up to 30% of women over 30 years of age, and are the most common cause of hysterectomy in premenopausal women. Leiomyomas frequently proliferate at a greater rate during pregnancy and can be extremely problematic. An examination of the basic cellular processes involved in myometrial function often requires the use of human myometrial cells in primary culture. However, these cells can be used only for several passages, and the magnitude of many responses changes with time in culture. Many laboratories do not have ready access to clinical material, and it would be essential to have immortalized human myometrial cells with all the properties of a low-passage phenotype. Transformed cells tend to be genetically unstable, as they can be polynucleated and can lose or gain chromosomes. However, Clontech, in collaboration with Geron Corp., has introduced several telomerase-immortalized cell lines that have the extended lifespan of transformed cells, but unlike transformed cells, maintain the appearance and behavior of normal primary cells. The purpose of this project is to develop telomerase-immortalized cell lines from term-pregnant human myometrium to eliminate variability between passage numbers and allow genetic manipulations of myometrial cells to fully characterize signal pathways. During this grant period we will: (1) immortalize early passage human myometrial cells in primary culture by infection with a retroviral vector expressing human telomerase (hTERT, obtained by material transfer agreement from Geron Corp.) and select clonal cell lines by puromycin resistance; and (2) examine maintenance of primary culture phenotype in immortalized cells by a number of criteria, including retention of very specific signal pathways involved with several different classes of agonists. Karyotyping will also be performed to ensure the lack of chromosomal alterations in immortalized cells. Criteria to be examined include alpha-actin immunocytochemistry, Akt phosphorylation in response to insulin treatment, stimulation of myometrial cell growth by lysophospholipids, quantification of oxytocin receptor ligand binding sites and upregulation by lysophosphatidic acid, oxytocin stimulation of RGS2 mRNA expression and ERK phosphorylation, EGF-stimulated phosphorylation of the EGF receptor, transactivation of EGF receptors by oxytocin, myosin light chain phosphorylation in response to uterotonic agents, and analysis of a number of signal pathways using gene array analysis.